Expression Level of a Phenylalanine Ammonia-Lyase Gene in Poinsettia Is Negatively Correlated with Poinsettia Branch-Inducing Phytoplasma Titer

ABSTRACT Poinsettia is an important ornamental cultivated worldwide. Commercial poinsettias are almost universally infected with a pathogen known as the poinsettia branch-inducing phytoplasma (PoiBI), which can increase the level of branching in host plants and make the plants more desirable to consumers. Despite PoiBI’s crucial role in poinsettia production, little is known about PoiBI-poinsettia interactions in regard to the pathogen’s in planta population dynamics. The expression profiles of a phenylalanine ammonia-lyase gene (Euphorbia pulcherrima PAL [EpPAL]) and the PoiBI titers in poinsettia tissues were investigated. Differential gene expression analyses using quantitative PCR (qPCR) showed that EpPAL expression levels differed significantly across tissue types. The highest expression levels were detected in stems, followed by root. Lower EpPAL expression levels were detected in leaf tissues, particularly in source leaves closer to the base; the average expression level in these leaves was only one-seventh of that detected in stems. Phytoplasma concentrations in source leaves close to the base were significantly greater than the other tissue types; the average value was 7.6-fold of that detected in stem tissues, which had the lowest phytoplasma titers. A negative correlation between EpPAL expression level and PoiBI load was detected, suggesting that the products of EpPAL-associated pathways or other genes indirectly associated with EpPAL may interfere with PoiBI’s growth. While additional studies are needed to validate these interpretations, the results from this work provide new insights into PoiBI-poinsettia interaction and showed that correlations between pathogen load and defense-related genes could be detected in phytoplasma-associated pathosystems. IMPORTANCE Phytoplasma-plant interactions are interesting subjects for fundamental and applicative research. Although many studies have characterized molecular interplays between these pathogens and hosts, knowledge on relationships between phytoplasmas’ in planta population dynamics and host gene expression remains scarce. By using the poinsettia branch-inducing phytoplasma (PoiBI) and poinsettia as a model system, a negative correlation was observed between the expression level of a plant defense-related gene and the pathogen’s titer. The findings provide potential explanations to PoiBI’s distribution patterns in the plant and highlight the importance of studying phytoplasma-plant interactions in regard to the pathogen’s population dynamics in other pathosystems.

2 NanoDrop One. The samples were stored at -20°C until use.
Generation of plasmid-based standard curves for qPCR assays. Prior to the qPCR tests, the target amplicons were amplified with conventional PCR. For both EpPAL and EpEF, the assays were conducted with the GoTaq Green Master Mix (Promega). Each 20-μl reaction included 0.4 μM of each primer (Table S1) and 1 μl of 10-fold diluted cDNA. The cycling conditions were 2 min at 95°C, 35 cycles of 30 s at 95°C, 30 s at 60°C, 1 min at 72°C, followed by 10 min at 72°C. The amplicons were cloned with the pGEM-T Easy Vector System (Promega). Plasmids carrying the target gene fragments were extracted from Escherichia coli JM109 transformants using a QIAprep Spin Miniprep Kit (Qiagen) and digested with PstI-HF (New England Biolabs). Linearized plasmids were recovered using QIAquick Gel Extraction Kit (Qiagen), quantified using NanoDrop One and diluted.
Whenever qPCR assays were conducted, the plasmids were serial-diluted and tested along with the plant DNA samples. To assure that the qPCR results were comparable among different runs, the standard curves for each gene in each set of data were constructed using aliquots of the same preparation of linearized plasmids.
Differential gene expression assays. For the gene expression assays, all qPCR runs were completed using the iQ SYBR Green Supermix and a CFX Connect Real-Time PCR Detection System (Bio-Rad). Quantification of EpPAL copy numbers was achieved using primers PAL1F and PAL1R (Table S1) designed in a previous study (1). The assays were conducted in 20-μl reactions including 0.4 μM of each primer (Table S1) and 1 μl of cDNA.
The qPCR conditions were set as previously described (1). For EpEF, primers EpEFfwd and EpEFrev (Table S1) developed in a previous work (2) was used. The 20-μl qPCR reactions for these tests contained 0.25 μM of each primer and 1 μl of cDNA. The qPCR conditions 3 were 3 min at 95°C, 40 cycles of 10 s at 95°C, 10 s at 58°C, 30 s at 72°C, followed by a dissociation curve assay. Each cDNA and plasmid DNA sample had three technical replicates and all assays included no-template controls. Differential gene expression analysis was conducted using the delta-delta-Ct method (3)  The 16S rRNA gene's copy numbers were determined with TaqMan assays using primer set CYS2Fw/CYS2Rv and the probe CYS2Probe (Table S1) designed in a previous study (4).
SYBR green assays using primers PAL1F and PAL1R (Table S1) were conducted to determine EpPAL's copy numbers. Similar to the gene expression analyses described above, PCR was conducted prior to the qPCR assays, and the target amplicons of both genes were cloned; assays using primer pair CYS2Fw/CYS2Rv and poinsettia DNA were conducted as previously described (1); assays using PAL1F and PAL1R were conducted as described above (using plant DNA instead of cDNA as templates). After cloning the amplicons, the plasmids were extracted, linearized, purified, and diluted to 5 ng/μl. Serial-diluted plasmids were tested along with the plant DNA samples, allowing absolute quantification of the genes' copy numbers (5).
The amplification conditions for the qPCR tests were set as previously described (1), and 10 ng of template DNA was used for each reaction. The CFX Connect Real-Time PCR Detection System (Bio-Rad) was again used for these assays. The TaqMan assays were completed using the iTaq Universal Probes Supermix (Bio-Rad), while the SYBR green tests were conducted with the iQ SYBR Green Supermix (Bio-Rad). Each plant DNA and plasmid 4 DNA sample had three technical replicates and all runs included no-template controls. The copy numbers of the two genes in the tested samples were calculated based on the standard curves as previously described (5,6).
Because previous studies reported that phytoplasmas have two copies of 16S rRNA gene in their genomes (7), the 16S rRNA gene's copy number of each sample was first divided by 2; the resulting value was then normalized (divided) by EpPAL's copy number.
Data analysis. Only data generated from qPCR runs with PCR efficiencies above 87.2% (R 2 > 0.99) were used for statistical analysis. Two-way analysis of variance [ANOVA; "tissue type" x "plant difference" (random factor)] tests without interaction were conducted on EpPAL expression (delta-Ct) and PoiBI titer (phytoplasma concentration) values. Post-hoc tests were conducted with the Fisher's least significant difference (LSD) procedure. The association between EpPAL expression levels (delta-Ct) and PoiBI titers were examined using a Spearman's rank-order correlation test.